The prophenoloxidase activation pathway in insects is an integral component of the host immune system against microbial infection. Phenoloxidase (PO) catalyzes the production of quinones that are precursors for cuticle sclerotization, wound healing, and melanotic encapsulation of invading microorganisms. Due to high reactivity and cytotoxicity of quinones, proteolytic activation of prophenoloxidase (PPO) has to be tightly regulated as a potent, local reaction against nonself. In insect vectors of human diseases, initiation and control of this process may be interfered by proteins from pathogens or parasites. Knowledge of the activation process from biochemical model insects such as Manduca sexta will be useful for understanding and manipulating a similar system in blood-feeding insects that transmit human diseases. Three PPO-activating proteinases (PAPs) isolated from M. sexta require cleaved serine proteinase homologs (SPHs) for PPO activation. Molecular cloning and sequence comparison indicate that the PAPs and SPHs all contain regulatory clip domains. We plan to expand our current research on PAPs to acquire in-depth understandings of the molecular mechanisms that trigger and regulate the PPO activation in M. sexta. The specific aims of this project are: 1) Characterization of HP14, an initiation proteinase of the PPO activation cascade; 2) Isolation and cDNA cloning of an activating enzyme for proSPH-1 and proSPH-2; [unreadable] 3) Functional analysis of the clip domains in PAP-2. [unreadable] [unreadable]